Our projects aim at understanding the complex signaling networks induced by type I interferons (IFN-α/β). What distinguishes these cytokines from other helical cytokines is the existence of a multiplicity of IFN subtypes that bind to the same ubiquitous receptor and the extreme pleiotropy and cell specificity of the biological responses. By studying receptor dynamics in response to different subtypes and in distinct cellular backgrounds, we hope to unravel molecular mechanisms underlining the pleiotropic responses to the IFNs. The ongoing functional and biochemical characterization of novel Jak-interacting proteins will broaden our understanding of signaling pathways initiated by cytokines and may also reveal additional functions of Jak enzymes.

IFN-α/β in the control of human CD4+ T lymphocyte homeostasis (E. Dondi)

Upon infection, IFN-α/β is rapidly secreted and participates in building up the early innate response. IFN-α/β also modulate adaptive immune responses, notably T cell-mediated responses, indirectly via its effects on dendritic cells. We sought to characterize the direct impact that IFN-α/β have on human T lymphocytes and to this end we measured proliferative and apoptotic signals in naïves T CD4+ T cells purified from umbilical cord blood, before and after their in vitro activation. We found that IFN-α protects T cells from mitochondrial-dependent apoptosis early upon TCR/CD28 activation. IFN-α acts by delaying entry of cells into the G1 phase of the cell cycle as well as by modulating proteins of the Bcl2 family. We also found that, at later time post-activation, T cells exposed to IFN-α showed increased levels of surface Fas associated with partially processed caspase-8, a key component of the extrinsic apoptotic pathway. Overall, our findings support a model whereby IFN-α favors clonal expansion, yet it primes cells to antigen-induced cell death occurring at the end of an immune response. This study further demonstrates that IFN-α exerts different effects according to the activation state of the T lymphocyte.

Dynamics and traffic of the type I IFN receptor: a role of the Jaks (J. Ragimbeau, Z. Marijanovic)

The four members of the mammalian Janus or Jak family of tyrosine kinases are non-covalently associated with cell surface receptors of helical bundled cytokines. In the heterodimeric type I IFN receptor, Tyk2 associates with the IFNAR1 subunit and positively influences its plasma membrane expression. We are interested in the intracellular fate of the IFN receptor subunits following ligand binding and in the role that Jak proteins play in post-binding events. Binding of IFN-α stimulates internalization of IFNAR1, a process that requires its serine phosphorylation and the recruitment of a ubiquitin E3 ligase. We have studied the dynamics of IFNAR1 upon ligand stimulation, with the aim to determine whether Tyk2 regulates it at any step. Using various approaches, we have shown that IFN-induced internalization of IFNAR1 does not require active Tyk2. Conversely, Tyk2 kinase activity is required to efficiently direct ligand-engaged IFNAR1 towards the lysosomal compartment and we are presently looking into the precise role exerted by Tyk2 in this context. The traffic of the other subunit of the IFN receptor (IFNAR2) is also being analyzed. Particular attention will be given to analyses of the receptor dynamics in different cell types and in response to two IFN subtypes (α2 and β), as we think that these aspects may critically account for the pleiotropic responses to the IFNs.

Study of Jamip1 in T lymphocytes (V. Libri and G. Pizza)

We identified Jamip1 (Jak and microtubule interacting protein) for its ability to bind to the FERM homology domain of Tyk2. Jamip1 belongs to a novel family of three genes conserved in vertebrates and predominantly expressed in neural tissues and lymphoid organs. These proteins lack known domains and are rich in coiled coils. Jamip1 comprises a N-ter region that targets the protein to microtubule polymers and, when overexpressed in fibroblasts, profoundly perturbs the microtubule network. The C-ter domain interacts with Jak kinases and can be target of multiple phosphorylation. The restricted Jamip1expression in lymphocytes and neurons, and its association with the microtubule cytoskeleton suggest a specialized function in dynamic processes such as cell polarization, local segregation of molecules or complexes, and/or in trafficking of secretory organelles/endocytic vesicles along microtubule tracks towards the immune synapse in lymphocytes and from/towards the neuronal synapse. We are interested in elucidating the function of Jamip1 in T lymphocytes. Activation of helper and cytotoxic T lymphocytes by antigen-presenting cells involves polarization of the lymphocyte towards the contact area and the formation of the immunological synapse. Cytoskeletal rearrangements are necessary for the stability of this interaction, for the maturation of the immunological synapse and for sustained T cell signaling. The microtubule organizing center (MTOC) reorients towards the synapse after TCR engagement and this may play a crucial role in the accumulation of TCR at the central synapse, the segregation of signaling proteins and the relocation of the secretory machinery that leads to polarized delivery of lymphokines, cytolytic granules. We are presently exploring the involvement of Jamip1 in early and late events occurring upon TCR stimulation of CD4+ T lymphocytes, by using an RNAi approach and by localizing fluorescently labeled Jamip1.

Study of Pot1, a novel interactor of Tyk2 (Milica Gakovic)

From a yeast two-hybrid screen using as bait the FERM-homology domain of Tyk2, a novel ubiquitously expressed gene was identifiedand provisionally named Pot1 (Partner of Tyk2). No close homologues are found in the human genome, but Pot1 encodes several isoforms. Pot1 is a novel Jak-interactor, involved in IFN signaling and associated to raft-containing microdomains. An exhaustive two-hybrid screening of highly complex human libraries is being performed in order to identify potential Pot1 interactors of known function. Subcellular localization studies will help to identify the nature of the cytoplasmic vesicles where Pot1 accumulates.